Friday October 11, 2019 from 08:00 to 08:30
Characterization of PERV virus particles in the CRISPR/Cas9 inactivated cell line PK15 clone 15
Antonia W. Godehardt1, Nicole Fischer1, Paula Rauch1, Barbara Gulich1, Klaus Boller1, George M. Church2, Ralf Toenjes1.
1Division of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany; 2Department of Genetics, Harvard Medical School, Boston, MA, United States
Introduction: PERV infecting human cells in vitro is a major objection for conducting XTx clinical trials. CRISPR/Cas9 technology enabled for the first time the generation of living pigs, free of functional PERV (1, 2). However, the susceptibility of these animals and medicinal products derived thereof towards reinfection with PERV remains unclear. In this study we characterized a CRISPR/Cas9, PERV inactivated cell line (PK15 clone 15) and its susceptibility to exogenous, infectious PERV as a representative model for genetically engineered porcine tissue and cells.
Methods: Cells basal expression of PERV pol and the porcine PERV-A receptor (POPAR) as well as its reverse transcriptase (RT) activity was determined. Subsequently, PK15 clone 15 cells have been inoculated with virus containing cell free supernatants for 24h. The virus inoculum was either derived from PK15 producer cells for PERV-A and -B, from HEK 293FT/B(33)ATG producer cells for PERV-B (3), from ST-IOWA/PERV-C (1312) or -(5683) producer cells for PERV-C (4) and from HEK 293/5° producer cells for PERV-A/C (5, 6). Parameters such as RT activity, virus expression and virus release were monitored weekly for 55 days. Analyses were performed by enzymatic RT activity assay as well as one-step qRT-PCR for RNA expression. In addition, cells and supernatants were analyzed by Western Blot for presence of viral capsid protein p30. Formation of viral particles was imaged by electronmicroscopy and immunofluorescent staining. Infectivity of the supernatant of PK15 clone 15 cells was tested in a downstream infection assay using swine testis-IOWA (ST-IOWA) cells as target.
Results: Our data indicate that PK15 clone 15 cells still produce viral proteins assembling to impaired viral particles. The virions show an irregular morphology that diverges from mature wildtype particles. The capsid is less dense and the particles are no longer infectious as tested in a downstream infection assay using supernatant of PK15 clone 15 to infect susceptible ST-IOWA cells. To exclude that the missing susceptibility to reinfection, especially to PERV-A, is affected by viral host receptor(s) the expression of POPAR was quantified. PK15 and PK15 clone 15 cells do equally express POPAR. In conclusion, PERV particles produced by PK15 clone 15 are impaired, not suitable to induce productive infection. While constitutively expressing PERV PK15 clone 15 cells are protected against PERV superinfection.
Conclusion: Besides hyperacute and delayed rejection the microbial as well as virological safety such as infectious risk represented by porcine endogenous retroviruses (PERV) are eminent. Here we show that the CRISPR/Cas9 PERV inactivated cell line PK15 clone 15 is not susceptible to PERV re-infection (superinfection) while constantly producing impaired virions. The cells express viral pol sequences but do not show viral RT activity. As such, they are no longer capable for productive infection.
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